TULANE COBRE: PHOSPHORYLATION IN SOLID MUSCLE TUMOR ALVEOLAR RHABDOMYOSARCOMA

肺泡横纹肌肉瘤（Alveolar Rhabdomyosarcoma， ARMS）是儿童最常见的软组织肉瘤之一，其特征是t(2;13)（q35;q14）染色体易位，导致两种对肌肉发育重要的转录因子Pax3和FKHR融合。目前，调控Pax3和致癌融合蛋白Pax3- fkhr活性的潜在分子机制以及这种调控在ARMS发展中的重要作用尚不清楚。因此，本提案的总体目标是了解磷酸化如何调节Pax3的转录活性，以及Pax3- fkhr如何改变这种调节。这一目标将通过两个具体目标来实现：(1)研究磷酸化在Pax3和Pax3- fkhr调控中的作用。这个特定的目标将通过确定Pax3和磷酸化位点来解决

The solid muscle tumor Alveolar Rhabdomyosarcoma (ARMS), one of the most frequent soft tissue sarcomas in children, is characterized by the t(2;13)(q35;q14) chromosomal translocation, which results in the fusion of two transcription factors important for muscle development, Pax3 and FKHR. At present, little is known about the underlying molecular mechanisms regulating the activities of Pax3 and the oncogenic fusion protein Pax3-FKHR and how this regulation may be important for the development of ARMS. Therefore, the overall objective of this proposal is to understand how phosphorylation regulates the transcriptional activity of Pax3 and how Pax3-FKHR may alter this regulation. This objective will be addressed through two specific aims: (1) To investigate the role of phosphorylation in the regulation of Pax3 and Pax3-FKHR. This specific aim will be addressed by identifying the sites of phosphorylation on Pax3 and Pax3-FKHR, determining how phosphorylation affects the molecular biological activities of each protein (i.e. - DNA-binding, protein stability, and transcriptional activity), determining how phosphorylation alters the Pax3- and Pax3-FKHR-dependent enhanced proliferation and differentiation of primary myoblasts, and analyzing the importance of phosphorylation on the oncogenic activity of Pax3-FKHR. (2) To identify the kinases responsible for phosphorylating Pax3 and Pax3-FKHR. This specific aim wil'. be addressed by using standard chromatographic techniques to isolate the kinases that phosphorylate Pax3 and Pax3-FKHR from primary myoblasts, confirming that these kinases are responsible for the phosphorylation in vivo, and utilizing kinase inhibitors to determine how inhibition of these kinases effects the previously described Pax3 and Pax3-FKHR-dependent biological effects (i.e. - enhanced proliferation, enhanced apoptosis, and oncogenic activity). The comparison of the differences in the regulation of Pax3 and Pax3-FKHR by phosphorylation will provide important insights into the role of Pax3-FKHR in the formation of ARMS.